biomolecules Article Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro Julia Hohenbichler 1 , Georg Aichinger 1 , Michael Rychlik 2 , Giorgia Del Favero 1 and Doris Marko 1,* 1 Department of Food Chemistry and Toxicology, University of Vienna, 1090 Vienna, Italy; [email protected] (J.H.); [email protected] (G.A.); [email protected] (G.D.F.) 2 Chair of Analytical Chemistry, Technical University of Munich, 80333 Munich, Germany; [email protected] * Correspondence: [email protected] Received: 18 June 2020; Accepted: 30 June 2020; Published: 9 July 2020 Abstract: Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin-O-deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract’s potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins’ efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells. Keywords: emerging mycotoxins; combinatory effects; synergism; phase I metabolism 1. Introduction The fungal species Alternaria alternata is known to infest crops including cereals, fruits, and vegetables [1,2]. A variety of Alternaria species have been reported to produce more than 70 secondary metabolites that might co-occur in food and feed. The chemical composition of the resulting highly diverse mixtures depends on several growth conditions [3]. Out of the most prevalent metabolites, several have been characterized chemically and were demonstrated to act as mycotoxins. Thereof, the two dibenzo-α-pyrones alternariol (AOH) and alternariol monomethyl ether (AME) are among the most important Alternaria toxins with respect to occurrence [2,4]. Contamination studies on the emerging Alternaria mycotoxins such as the perylene quinones altertoxin I (ATX-I) and altertoxin II (ATX-II) are still scarce (Figure1a,b); yet, in light of their mutagenic potential, they might pose an underestimated health risk [4–6]. Frequent crop infestation by Alternaria spp., concomitant with simultaneous production of various secondary metabolites, as well as chemical stability during food processing and storage, results in persistent co-occurrence of Alternaria toxins [2,4,7–9]. A call by the European Food Safety Authority to collect further data on Alternaria toxins was followed Biomolecules 2020, 10, 1018; doi:10.3390/biom10071018 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1018 2 of 16 by a rise in studies investigating nutritional exposure [1,2]. These uncovered co-contaminations of various Alternaria toxins in sunflower seed oil, tomato products, infant foods, and diverse fruit and vegetable juices [10–12]. Recently, a low abundance of AOH and AME was reported even in breast milk samples [13]. Figure 1. Chemical structures of (a) ATX-I, (b) ATX-II, (c) AOH and (d) AME. The two dibenzo-α-pyrones AOH and AME (Figure1c,d) were previously reported to a ffect cell viability [14] and to moderately induce DNA strand breaks [15]; the latter occurring upon poisoning of topoisomerase II, as demonstrated for AOH [16]. The role of the oxidative stress inducing potential of both dibenzo-α-pyrones towards their genotoxic potential still remains unclear [17,18]. Besides, AOH was reported to exhibit cytotoxic [8], clastogenic [19], and immunomodulatory properties [20–23] in vitro as well as fetotoxic [24] capacities in vivo and to possess estrogenic as well as other endocrine disruptive potentials [19,25]. The perylene quinone ATX-II was previously shown to exert genotoxic capacities [26], alter cell membrane biophysical properties of intestinal cancer cells [27], and inhibit the NF-κB pathway in THP-1 derived macrophages [28]. Both ATX-I and ATX-II were reported to possess mutagenic activity in S. typhimurium, with a substantially lower potency for ATX-I [5], while the nitrosylation of ATX-I in turn was found to increase mutagenicity against two strains of Salmonella [29]. Considering a previous report of ATX-II to be de-epoxidized yielding ATX-I in vivo [30], both substances should be implemented in toxicological investigations for a proper risk assessment. The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix Per-Arnt-Sim transcription factor, can be activated via ligand binding [31]. Upon activation and translocation into the nucleus, the ligand bound to AhR heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). This complex regulates the transcription of the cytochromes P450 (CYP) 1A1, 1A2, and 1B1, enzymes involved in xenobiotic phase 1 metabolism, via interaction with the xenobiotic responsive element [32,33]. Additionally, AhR activation has been shown to affect cancer cell proliferation, cell migratory capabilities, and therefore also crucial steps in metastasis and innate as well as adaptive immunity [34,35]. Besides, several CYP isoforms are frequently induced by their own substrates. Hence, prominent ligands of the AhR behave also as inducers of CYP 1A1 activity, such as 2,3,7,8–tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (B[a]P), which are suggested to be involved in increased breast cancer risk [36–40]. Furthermore, distinctively elevated CYP 1A1 and CYP 1B1 protein expression as well as EROD activity were previously reported for breast cancer tissues in comparison to adjacent non-tumor tissues [41]. Extensive research has uncovered a complex crosstalk between AhR signalling and estrogen receptor (ER) related pathways in estrogen-sensitive tissues. Diverse mechanisms have been proposed to take part and lead to disparate effects of activation of AhR as well as ER [42–45]. A standard method used to determine CYP 1A1 enzyme activity induction upon exposure to AhR ligands is the 7-ethoxyresorufin-O-deethylase (EROD) assay [46]. The dealkylation of 7-ethoxyresorufin (7-ER) yielding the fluorescent substance resorufin was shown to be primarily exerted through CYP 1A1, followed by CYP 1A2 and CYP 1B1 [47]. AOH and AME were reported to serve as substrates for different CYP enzymes, particularly for CYP 1A1, resulting in the generation of hydroxylated metabolites [48,49]. Previous studiesuncovered the induction of CYP 1A1 expression to be AhR dependent for both dibenzo-α-pyrones in murine Biomolecules 2020, 10, 1018 3 of 16 hepatoma cells and AOH in human esophageal cancer cells [50,51], whereas the latter turned out to be irrelevant for the genotoxic capabilities of AOH. Although data on the metabolism of altertoxins is scarce, ATX-II was reported to induce CYP 1A1 activity and raise CYP 1A1 transcript levels in KYSE 510 cells [51]. The co-occurrence of toxins may lead to interactions apart from additive combinatory effects, such as positive effect amplifications (synergism) or a reversing impact (antagonism) [52]. A previous combinatory study of AOH and AME revealed a synergistic effect towards cytotoxicity in Caco-2 colon carcinoma cells [53]. Recently, complex Alternaria extracts were found to exert anti-estrogenic capacities towards Ishikawa endometrial cancer cells that could partially be ascribed to an ER-AhR/ARNT complex crosstalk [54]. Hence, this study aimed to uncover the potential of naturally occurring mixtures of Alternaria toxins to enhance CYP 1 activity and their combinatory effects in this respect applying the EROD assay. For this purpose, two variants of an Alternaria extract, the first onward named “complete extract” (CE) gained from inoculation of rice with the Alternaria alternata strain DSM62010 [55] and further stripped from ATX-II and STX-III to obtain the second variation named “reduced extract” (RE) [54] were applied. Further, the CYP 1 activating potential of single Alternaria toxins, as well as binary mixtures according to the complex extracts, was investigated utilizing the estrogen-sensitive mammary adenocarcinoma cell line MCF-7. Apart from their pronounced genotoxic properties [26], in vitro data on ATX-I and ATX-II and their diverse bioactive mechanisms and interactions with co-occurring mycotoxins for proper risk assessment is still lacking. Thus, the present study was performed with special emphasis on the perylene quinone derivatives and their effects in combinations. 2. Materials and Methods 2.1. Chemicals and Reagents Cell culture media and supplements were purchased from Gibco Thermo Fisher Scientific (Waltham, MA, USA) and Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Labware for experiments was obtained from Sarstedt